High density Tiling array analysis of Streptococcus pneumoniae TIGR4 genome

The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure. S. pneumoniae strain TIGR4 (ref) was grown in THYmedium, Todd-Hewitt broth supplemented with 0.5% yeast extract. Cells were harvested from duplicate cultures from mid-log phase (OD600 nm, 0.4–0.6) of growth by centrifugation. The harvested pellets were washed twice in sterile PBS (pH 7.0) and stored at -80°C. RNA was purified from frozen bacterial pellets using Qiagen RNeasy kit by following the manufacturer’s protocol. Isolated RNA was treated with DNase to remove genomic DNA contamination, and purity was checked by performing a one-step RT-PCR using primers specific for 16S rRNA in presence or absence of reverse transcriptase. RNA concentration and quality were determined by using Agilent Bioanalyzer. Purified RNA was stored in nuclease free water at -80°C. One microgram of total RNA was used by Nimblegen systems for labeling and hybridization. 2 biological replicates (single colored) were used in the analysis. 20,000 random probes were designed on the chip as a negative controls to measure non-specific hybridization. 35 genes identified through proteomics of same sample were used as positive controls.